As the site of complex cellular interactions and receptor mediated signaling events that lead to the production of mature T cells, the thymus is a likely target for mutations that result in primary immunodeficiency. Although many cell surface markers are available to delineate the stages of T cell development in the thymus, we know very little about the underlying molecular events that regulate these processes. This is particularly true for positive selection, the differentiation of developing thymocytes as a consequence of T cell antigen receptor (TCR)- mediated activation. We have assembled a unique set of tools and offer a unique approach to narrow the search for genes that play a critical role in positive selection. Rather than attempt to isolate all genes whose expression is associated with a particular development stage of T cell maturation, our approach is designed to identify genes that are regulated by TCR activation of specific signaling pathways that are required for T cell development. In order to accomplish this, we will take advantage of the immature thymocyte cell line DPK, that we have previously isolated and characterized. These cells maintain the ability to differentiate in culture, and we have shown that this differentiation is both Ras and calcineurin dependent, as is positive selection of normal thymocytes. In addition, DPK cells that express active Ras show a partially differentiated phenotype. Utilizing suppression subtractive hybridization and DPK cells that expresses active Ras or a dominant negative mutant of Ras, we will isolate genes that are specifically induced by TCR activation of the Ras/MAP kinase pathway in double positive thymocytes. A similar approach is described for isolating genes that are specifically induced by TCR mediated Calcineurin activation. A strategy for screening these differentially expressed sequences for genes that are candidates to play an important role in T cell development is outlined. In vitro gene transgene transfer and transgenic and gene targeting strategies will ultimately be used to determine the function of these genes.